Preclinical Assays for Identifying Cancer Chemopreventive Phytochemicals

Dietary factors influence carcinogenesis in a variety of tissues. The consumption of fruits and vegetables is associated with a decreased risk of several types of epithelial malignancies. In addition, there are interrelationships between diet, environmental factors, and genetics that can affect cancer risk. Potential chemopreventive agents against cancer development can be found among nutritive and/or nonnutritive compounds in inedible and edible plants. To identify potential cancer chemopreventive agents, scientists are evaluating hundreds of phytochemicals for the prevention of cancer. This short review article describes in vitro and in vivo assays reported to identify potential cancer preventive compounds from plants.


Introduction
Cancer mortality rates in the developed countries have increased throughout this century.It is already the leading cause of death in some Western countries [1,2].In Japan, the progressive introduction of Western dietary habits, especially increased fat intake and reduced carbohydrate and dietary fiber intake, has increased the incidence of colon cancer and related deaths [3].Great advances have been made in the pharmacologic-based treatment of malignant epithelial neoplasms (cancers).In addition, there is a marked increase in the understanding of cell and molecular mechanisms underlying carcinogenic processes.However, therapy for advanced neoplastic disease remains limited.This may be due to the fact that advanced neoplasms contain a large number of genetic and molecular alterations that contribute to the maintenance of their neoplastic progression.
The chemopreventive approach against cancer development is highly attractive.Although highly attractive from a theoretical point of view, practical limitations may exist with respect to developing novel and effective chemopreventive agents.Most importantly, practical clinical endpoints are not definite.The efficacy of cancer chemoprevention can be determined by comparing the incidence of nondeveloped disease in a treated group to that of a control group.Such a clinical trial is labor intensive, very costly, and timeconsuming.In addition, practically such trials cannot be conducted for the rapidly increasing number of chemopreventive agents being identified.These considerations suggest the use of certain intermediate biomarkers as indicators of clinical efficacy.Validated intermediate biomarkers thus may allow relatively small chemopreventive trials to be conducted within a short-term period.However, there are no universal approaches to determine intermediate biomarkers and their method of validation has still not been proven.Some herbal and botanical products are likely to possess cancer preventive activities [4][5][6][7].Many cancer patients use complementary and alternative medicines, including phytochemicals in addition to, or following the failure of standard cancer therapy [8].The term phytochemical applies to any plant-based substance, but in the field of nutrition and cancer this term is usually applied to nutritive and nonnutritive chemicals that occur naturally in fruits and vegetables.A diet rich in fruits and vegetables has long been suggested to correlate with reduced risk of certain epithelial malignancies, including cancers in the lung, colon, prostate, oral cavity, and breast [4][5][6][7][9][10][11][12].Also, the cancer prevention potential of Mediterranean diets based mainly on olive tree products is known [13].The major component of the leaves and unprocessed olive drupes of Olea europaea is oleuropein and the majority of polyphenols found in olive oil or table olives are derived from its hydrolysis.Oleuropein is a novel, naturally occurring antioxidant compound, which may possibly be used to prevent cancer [14][15][16] and cardiotoxicity induced by doxorubicin [17].
Searching for medicinal benefits from edible or inedible plants is not a new idea since numerous modern medicines have plant origins.Given that the ingestion of some plant foods results in reduced risk for cancer, researchers are delving into the identification of phytochemicals with cancer preventive ability in studies in vitro (cell culture), in vivo (model animals) and those in humans [18].Phytochemicals can be roughly classified into four groups based on their mechanisms of chemopreventive action, as shown in Table 1.
Preclinical studies focus on the identification of possible cancer preventive agents, short-term pharmacology, and assessment of toxicity.Agents that are within tolerable safety limits in humans then moved clinical trials to test their efficacy.This review briefly summarizes the in vitro and in vivo assays used to discover possible new chemopreventive agents possessing novel mechanisms of action.Surely there are many more assay systems currently used in different laboratories to find candidate cancer chemopreventive agents and determine their efficacy.Because of limited space, only limited assays that might be useful for carcinogenesis and chemoprevention studies are herein introduced.public for the chemoprevention of CRC.Although NSAIDs are excellent chemopreventive agents in animal models for CRC, dietary aspirin does not inhibit azoxymethane (AOM)induced colon carcinogenesis in rats that received a nonhigh fat diet.Because of side effects of NSAIDs that affect both COX-1 and COX-2 expressions, specific COX-2 inhibitors (-coxibs) were introduced to obtain cancer chemopreventive ability without side effects [25,26].They are indeed effective for CRC development in animal studies [27].However, certain COX-2 inhibitors have been reported to increase the risk of ischemic heart diseases [28].
Many phytochemicals and/or botanicals are routinely tested for anti-inflammatory properties in the hope of finding new sources of medicines for the treatment of chronic inflammation and for the cancer chemoprevention.The plant world is apparently rich in sources of COX and/or inducible nitric oxide (iNOS) inhibitors that are involved in inflammation-related carcinogenesis [29].Several of these have been found to inhibit cancer development in certain animal models.Curcumin [30] is able to inhibit the COX activity [31] in the skin [32] and suppresses skin cancer development in mice [33], tongue [34] and CRC in rats [35].Tea [36,37], given to human volunteers in a clinical trial, could reduce PGE 2 activity in the rectal mucosa [38].Resveratrol [39] is a potent inhibitor of COX [40] and is an inhibitor in the mouse skin and rat mammary carcinogenesis models [41].Silymarin [42] a COX and iNOS inhibitor [43] was also found to inhibit skin [44,45], colon [46], tongue [47], and prostatic [45,48] cancer development.However, the cancer preventive ability of carnosol and ginger substances, both COX inhibitors [49,50], has not fully been investigated [51,52].Numerous phytochemicals thus remain to be tested in animal models for the most common cancers such as colon, mammary, prostate, and lung.Nobiletin [53][54][55][56], zerumbone [57,58], and garcinol [59][60][61] are potent anti-inflammatory compounds present in plants that inhibit carcinogenesis in different tissues.A recent study demonstrated the chemopreventive ability of zerumbone, which has been shown to possess antiinflammatory effects in mouse lung and colon carcinogenesis [62].These phytochemicals [63], for example, curcumin [31,64], resveratrol [65,66], silymarin [67], from plants have multifunctional effects, including COX-2 inhibition and anti-inflammatory function, on a variety of events that involved carcinogenesis [68][69][70].
Most phytochemicals with chemopreventive ability [71] have direct antioxidant activity but many can also induce oxidative stress within cells when applied at high doses [11,72].For example, a simple phenolic acid protocatechuic acid is a potential cancer chemopreventive agent in a variety of tissues [73], but it enhances tumor promotion and oxidative stress in female ICR mouse skin via enhancing a promoter-induced inflammatory responses and promotion by affecting tyrosinase-dependent oxidative metabolism of protocatechuic acid [74].Therefore, care should be paid for applying strong antioxidants to clinical use as a chemopreventive agent.However, recently anti-inflammatory, antioxidative chemopreventive phytochemicals targeting signal transduction mediated NF-2 related factor-2 (Nrf2), nuclear factor-kappaB (NF-κB), and activator protein (AP)-1 that are redox-sensitive factors have been highlighted [72].We recently have demonstrated that such a compound and melatonin effectively suppress inflammation-associated colon tumorigenesis [62,75].
Angiogenesis the formation of new blood vessels from existing vasculature has been associated with neoplasms.Angiogenesis is critical to the transition of premalignant lesions in a hyperproliferative state to the malignant phenotype, which leads to tumor growth and metastasis.The intensity of angiogenesis as assessed by counting of microvessels in neoplastic tissue acts as a prognostic factor for many solid tumors, including CRC [76,77].Similarly, expression of angiogenic growth factors is associated with prognosis of a variety of cancers [76,77].Angiogenesis enables tumors to grow larger than 1-2 mm in diameter, invade surrounding tissue, and metastasise.Angiogenesis is already targeted by chemopreventive agents at various stages of drug development or in clinical practice [76,77].The biomarker measured in chemoprevention must have the potential for modification by therapeutic interventional agents.In this regard, angiogenesis is a particularly attractive biomarker.There are in vivo and in vitro assays using human endothelial cell neoplasms [78], umbilical vein endothelial cells [79] and for screening potential anti-angiogenic effects of candidate chemicals.Potentially nontoxic anti-angiogenic dietary compounds include green tea polyphenols, genistein, curcumin, resveratrol, linoleic acid, hesperidin, naringenin, and allyl disulfide [80].
The defect in apoptosis mechanism is recognized as an important cause of carcinogenesis [5,6].A dysregulation of proliferation alone is not sufficient for cancer development as suppression of apoptotic signaling is also required.Cancer cells acquire resistance to apoptosis by overexpression of anti-apoptotic proteins and/or by the downregulation or mutation of pro-apoptotic proteins.Various studies indicate that dietary constituents, particularly phytochemicals, can modulate the complex multistage process of carcinogenesis by several mechanism(s), including apoptosisinducing effects [81].They include (−)-epigallocatechin gallate, curcumin, genistein, indole-3-carbinol, resveratrol, isothiocyanates, luteolin, lycopene, caffeic acid, apigenin, sylymarin, gingerol, and capsaicin [81].

Mechanistic Screening Assays for Detecting Potential Chemopreventive Compounds
Potential chemopreventive agents are systematically screened in a battery of short-term assays which determine the inhibition or induction of biochemical and molecular processes involved in carcinogenic processes.As summarized in Table 2, these mechanistic-based assays are roughly divided into three major categories: (i) antimutagenesis assays which evaluate carcinogenesis blocking activities, (ii) antiproliferative and antiprogression screening assays, and (iii) assays assessing antioxidant and anti-inflammatory mechanisms.
In addition to these assays, studies are in progress to establish assays utilizing DNA microarray and proteomics to facilitate the discovery of new chemopreventive agents and novel molecular mechanisms of action [82,83].Such new emerging technologies allow the screening and monitoring of the expression levels of thousands of genes simultaneously [84].More importantly, the technology to make customized gene chips with specific genes is also possible.In addition to monitoring the alterations in gene expression patterns in tissues undergoing carcinogenesis, these chips can be utilized to evaluate subjects at risk, such as those carrying specific germline mutations and genetic polymorphisms [85].

In Vitro Efficacy Model Systems
Besides the mechanistic assays mentioned above, the systematic evaluation of cancer preventive agents includes screening compounds in short-term in vitro screens which aim to select agents for subsequent whole animal testing with insight into potential mechanisms of action.Use of primary cultured cells without aneuploidy is ideal because they possess relatively intact drug metabolizing systems and normal gene numbers.Epithelial cells used are rat tracheal epithelial cells, human lung cells, hyperplastic alveolar nodules in mouse mammary gland organ cultures, JB6 epidermal cells, and human foreskin epithelial cells.
In each assay, the substances are tested over a wide range of concentrations to determine EC 50 values.The assays include (i) a rat tracheal epithelial cell (RTE) assay which measures the ability of candidate chemopreventive agents to block the benzo[a] pyrene (B[a]P)-induced transformation of primary RTE cells [86]; (ii) an anchorage independence assay which is an effective method for detecting compounds that block carcinogenesis in the postinitiation stages and evaluates inhibition of anchorage independence in human lung tumor (A427) cells [87]; (iii) a mouse mammary gland organ cultures (MMOCs) assay which assesses the inhibitory activity of test chemicals on the development of carcinogeninduced hyperplastic alveolar nodules (HANs) in MMOC [88].This assay is similar in appearance to the alveolar nodules produced in mouse mammary glands in vivo [89]; (iv) an in vitro assay for antipromoters or antiprogressors which is designed to identify chemopreventive agents effective in the promotion or progressive stages of carcinogenesis in JB6 epidermal cells [90]; and (v) a human foreskin epithelial assay which determines the inhibitory potential of chemopreventive agents in blocking cell growth stimulation induced by the carcinogen propane sulfone [91].

In Vivo Short-Term Screening Assays
This type of short-term assays identifies agents that might block or arrest carcinogenesis in the early stages.Two experimental models, which reflect major cancers in humans, are being used.They include the rat and mouse colorectal aberrant crypt foci (ACF) assay [11,12,92,93] and a rat model of breast ductal carcinoma in situ model (DCIS) [94].

ACF Assay.
The ACF assay is a short-term model which can identify agents that may be effective in preventing CRC [11,12,92,93].ACF, which were first described by Bird [95], are putative preneoplastic lesions consisting of aggregates of single and multiple crypt cells that exhibit hyperplasia and/or dysplasia and are thought to be the earliest detectable lesions of CRC [96][97][98][99].Two different protocols have been developed: one which identifies compounds that inhibit initiation and a second treatment schedule which evaluates potential chemopreventive agents during the postinitiation phase of colorectal carcinogenesis.Details of these regimens have been described previously [11,12,100].In the former (the initiation protocol), rats are given a test agent in the diet one week prior to the administration of a colonic carcinogen, such as AOM and continuing throughout the five-week study period.In the latter regimen (the postinitiation protocol), rats are first treated with AOM, followed four weeks later by a test agent, which is given for additional weeks.Animals are sacrificed and the ACF frequency is determined by microscopic evaluation.

DCIS Assay.
The DCIS assay provides both toxicity and efficacy data for identifying candidate chemopreventive agents prior to testing in common mammary carcinogenesis models.Therefore, the induction of mammary tumorigenesis is initiated in weanling female SD rats by the intraperitoneal (i.p.) injection of the carcinogen N-methy1-N-nitrosourea (MNU) [94].In general, test agents are administered in the diet, starting one week after the carcinogen administration, and thereafter are continued until the termination of the study (45-50 days later).Mammary tissue specimens are excised and processed for histopathological analysis.The efficacy is estimated as the percent reduction in the number of DCIS lesions in comparison to controls that receive a carcinogen alone.

Animal Efficacy Assays
The  3.

Head and Neck Carcinogenesis Models.
Several wellestablished models of oral and respiratory tract cancer have been developed.In the hamster buccal pouch model [101], the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) is topically applied over a 12-week period, thus resulting in buccal pouch squamous cell carcinomas [102].Rats [4] and mice [103] develop tongue cancers when exposed to 4-nitroquinoline 1-oxide (4-NQO) [104].In rats, tongue squamous cell carcinomas and dysplasia can be induced by 4-NQO in drinking water (20 ppm) for 8 weeks [4].Tongue dysplasia occurs during 4-NQO treatment and the incidence of tongue squamous cell carcinoma is over 50% at 32 weeks after the exposure.In this model, test chemicals can be orally administered either before, during,  or following 4-NQO exposure [4].For the induction of lung adenosquamous cell carcinoma or squamous cell carcinoma of hamsters, MNU is administered over a 15-week period, thus resulting in approximately 40-50% of the treated animals after 6 months [105].Starting one week prior to MNU exposure, a test compound is administered over 180 days.In the second model, hamsters are given the carcinogen diethylnitrosamine (DEN), subcutaneously, twice per week over a 20-week period, resulting in the formation of lung adenocarcinomas in about 50% and tracheal tumors in over 90%, of treated animals [106].As in the MNU model, test agents are given prior to carcinogen exposure.The tobaccospecific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) can induce lung tumors in A/J mice and a large number of compounds are now available that inhibit the mouse lung tumorigenesis induced by NNK [107].Another lung cancer bioassay is the strain A mouse model [108].Strain A mice spontaneously develop lung tumors as early as 3-4 weeks, with lung tumor incidences approaching 100% at 24 months of age.A/J mice are also utilized for studies on colon tumorigenesis because of their susceptibility to colonic carcinogens [109].[11,12,110,111].According to established protocols, 1,2-dimethylhydrazine (DMH), AOM, or methylazoxymethanol (MAM) acetate is administered intraperitoneally or subcutaneously, thus resulting in colorectal adenocarcinoma development within 32-40 weeks in either species.DMH is first activated to form AOM and then is metabolized by the liver to form MAM, the ultimate carcinogen, which is excreted via glucuronide conjugation.In the AOM induction model, a single or multiple (up to 3 times) subcutaneous dose of AOM in male F344 rats results in the occurrence of colorectal adenocarcinoma and adenoma in approximately 70% of treated animals by 40 weeks.Again, the test agents can be orally administered either before, during, or following carcinogen treatment [112].In comparison to rats, mice should receive multiple exposures of a colonic carcinogen to induce colonic tumors and tumor development needs long-term period.A mouse model recently established for colorectal carcinogenesis [113], in which different colonic carcinogens are followed by a colitis-inducing agent, dextran sodium sulfate (DSS), is quite useful to identify potential chemopreventive agents within a short-term period [68,114].

Mammary Carcinogenesis Models.
Chemopreventive efficacy against mammary gland carcinogenesis is routinely assessed by either the MNU-or DMBA-induced models [115].Both protocols utilize female SD rats and require that the carcinogen is given as a single dose at 50 days of age.In some instances, the carcinogen is administered to older animals (180 days) which is more representative of the human target population.Tumor incidences at 120 days after carcinogen treatment are similar, ranging from 80-100% in the DMBA protocol and 75-95% in the MNU model.However, the histological types of tumors induced by the two carcinogens are different.DMBA-induced mammary tumors are predominantly adenoma and fibroadenoma, with some adenocarcinoma, whereas MNU-induced mammary tumors are invasive adenocarcinomas.The chemopreventive activity of the test agents is determined by the percent reduction in tumor incidence or percent increase in tumor latency relative to controls treated with the carcinogen alone.These models produce hormonally responsive tumors.In addition to these chemically-induced mammary carcinogenesis models, several genetically engineered animals have been introduced to investigate breast carcinogenesis and evaluate the efficacy of candidate chemopreventive agents.They include a COX-2 overexpressing mouse model [116], a Rasdriven mouse mammary tumorigenesis model [117], and a HER-2/neu transgenic mouse model [118].In addition, mammary stem cell models can be used for studying how the hormonally regulated paracrine interactions influence stem cells and the stem cell niche during mammary carcinogenesis [119].Another interesting model system for understanding normal human breast development or tumorigenesis is the orthotopic xenograft model that has the potential to improve the understanding of crosstalk between tissue stroma and the epithelium as well as factors involved in breast stem cell biology tumor initiation and progression [120].

Urinary Bladder Carcinogenesis Models. Urinary bladder neoplasms are typically induced by the carcinogen N-butyl-N-(4-hydroxybutyI)nitrosamine (OH-BBN) which
can induce invasive transitional cell carcinomas morphologically similar to those found in humans [121,122].This carcinogen is given either intragastrically or in drinking water over an 8-week period to 50-day old BDF mice (C57BL/6 × DBS/2-F l ) or F344 rats, thus resulting in a 40-50% incidence of bladder tumor incidence at 180 days after OH-BBN treatment.Treatment schedules for a test agent administration are as described above [123].

Skin Carcinogenesis Models.
Agents effective in inhibiting skin carcinogenesis are identified in a two-stage skin carcinogenesis model utilizing DMBA and TPA, which are applied topically to the back skin of SENCAR or CD-1 mice [124,125].Both strains of mice are highly susceptible to skin tumor induction.Skin papillomas appear as early as 6 weeks postcarcinogen treatment, eventually progressing to squamous cell carcinomas by 18 weeks [126].Test agents are generally administered in the diet or in some experiments are topically applied according to several predefined treatment regimens.As to melanoma chmoprevention study, we should read an elegant review for new perspectives of this research area [127].In addition to in vitro screening assay [128], several genetically altered animal (mouse) models of melanoma [129][130][131][132], including hepatocyte growth factor/scatter factor (HGF/SF) transgenic mice [133][134][135][136], have been introduced for prevention [137] and biology [138] of this neoplasm.Also, a three-dimensional skin reconstruction model [139] is useful for determining the therapeutic efficacy of selected chemicals or drugs in cultured melanoma cells [140].While epidemiological studies suggest sunlight as an etiologic agent for the pathogenesis of melanoma, recent experimental investigations by the group Meyskens, Jr. indicated that elevation of reactive oxygen species follows from melanin serving as a redox generator [127] and this may be involved in the etiology and pathogenesis of cutaneous melanoma.Such findings will help to establish novel preventive and therapeutic approaches to this malignancy.

Transgenic and Gene-Knockout Animal Models
Animal models that mimic the specific characteristics of human carcinogenesis may prove to be a valuable resource in both evaluating chemopreventive efficacy and identifying appropriate biomarkers for measuring the chemopreventive activity.Transgenic and gene knockout mice that carry well-characterized genetic lesions predisposing them to carcinogenesis are appropriate models for chemoprevention testing (Table 4).Some of the best developed models include the multiple intestinal neoplasia (Min) mouse [141] and other strains possessing lesions in the Apc gene [142].The Min mouse carries an Apc mutation similar to that found in human familial adenomatous polyposis (FAP) patients.These mice are predisposed to develop predominantly small intestinal adenomas, but a few in the large intestine.By manipulating two or more carcinogenesis-associated genes, such as modifier genes, in a single animal, closer approximations of human carcinogenesis may be possible.Numerous colonic tumors develop in the large bowel in Min mice at 3 weeks after one-week-exposure of DSS [143], thus suggesting the importance of gene-environmental interaction in cancer development [68].It might be feasible to knock out p53 in an animal that already carries another tumor suppressor defect such as Apc or p16.Recently, new transgenic animal models for mammary [144], tongue [145,146], pancreas [147], and gall bladder [148] cancers have been reported.These models might be useful for discovering possible novel cancer chemopreventive agents.

Combination Treatment
One strategy for improving the efficacy and lessening toxicity is using combinations of agents [149,150].Synergistic or additive effects may be observed when two agents with different mechanisms of action are combined [149].Such improved activity of inhibition may allow either or both of the agents to be given at lower doses, thereby reducing the toxic potential.Examples of chemical combinations producing positive results in experimental animal models include all-trans-N-(4-hydroxyphenyl)retinamide (4-HPR) and tamoxifen in the rat mammary gland [151] and 2dinuoromethylorinithine (DFMO) and piroxicam in the rat colon [152,153].The identification and evaluation of other effective agent combinations is an interesting and important research effort for chemopreventive agent development [154,155].Since different treatment schedules of the two drugs can be compared and evaluated for synergism, additivity, or antagonism using a quantitative method based on the median-effect principle of Chou and Talalay [156][157][158][159] can identify the ideal combination treatment for obtaining an improved effect in comparison to a single exposure of the potential chemopreventive agent [160][161][162].

Intermediate Biomarkers
Carcinogenesis proceeds through a very long preclinical period.The collective hope is that multiple opportunities exist for chemoprevention to arrest or reverse progression towards malignancy.Intermediate biomarkers of malignant neoplasms are the phenotypic, genotypic, and molecular changes that occur during carcinogenesis [69].An important component of chemopreventive agent development research is the identification and characterization of intermediate biomarkers that may serve as surrogate end points for cancer incidence reduction in chemoprevention clinical trials [163].Such efforts are critical to the progress of chemoprevention and have the potential for cost-effective development of chemopreventive research [5,6,69].Biomarkers include proliferation, apoptosis, growth factors and their receptors, genetic alterations, and so forth in the target tissues [162,164,165].In the hope of faster progress with fewer subjects and lower total cost, much effort is being expended on the search for reliable biomarkers to predict the likelihood of developing cancer and/or to signal the effectiveness of chemopreventive therapy [166].Considerable attention has been paid to identifying those markers that can act as surrogate markers for cancer development since favorable modulation of the surrogate end-point biomarker may demonstrate the effectiveness of a putative preventive treatment.However, the complexity of the biology challenges the ability to measure the effectiveness of attempts to arrest or reverse carcinogenesis, other than through costly and time-consuming prospective trials with the disease state as the endpoint.Despite much work, to date no prehistological biological or molecular intermediate marker has yet been validated for sporadic cancers.For chemoprevention of prostate cancer [167] and other types of cancer, natural and synthetic agents that may suppress, reverse, or regress precancer and delay progression to invasive cancer can be used [77].Epidemiological evidence suggests that environmental factors, such as diet, play a role in the development and progression of prostate cancer.The number of potential protective dietary compounds or whole dietary products that are indicated to have preventive effects is piling up and demands further evaluation.To face this scientific field, a strategy that combines prostate-specific antigen (PSA)-based clinical trials with experimental human xenograft studies to evaluate potential chemopreventive agents against prostatic cancer is proposed [168,169].PSA for prostate adenocarcinoma, even though has relatively poor specificity, is cheap and easily followed with minimally invasive procedures.

Future Direction
Epidemiologic evidence suggests that green tea polyphenols reduce the risk of some forms of cancer, while data for other commonly used phytochemicals is less convincing.Part of the problem lies in precise quantitation of foodborn phytochemicals where multiple dietary sources of a particular phytochemical are involved, as in recent studies of dietary quercetin on the prevention of heart disease.Future investigations may focus on specific foodborn phytochemicals and/or botanicals capable of intervening in critical pathways in carcinogenesis as indicated by the possible use of "natural" NSAIDs in the prevention of CRC and the soy-based phytoestrogens in the prevention of breast cancer.Much is to be learned by collaboration between cancer researchers and ethnopharmacologists for probing dietary prevention of cancer.In addition, the development and validation of new in vitro and in vivo assays of high predictive value for discovering agents with human chemopreventive potential is needed.Care, however, is needed when extrapolating in vitro data to in vivo models because it cannot be assumed that the effects seen when cells are exposed directly to active compounds that would be candidate chemopreventive agents will be seen when they are consumed in the diet.We should investigate whether they are capable of distribution throughout the body when they are absorbed after ingestion.The understanding of the molecular and cellular processes, such as gene and protein expression, apoptosis, angiogenesis, signal transduction, involved in carcinogenesis is rapidly increasing.Currently several translational studies using phytochemicals and bioactive compounds, such as indole-3-carbinol [170], ellagitannins [171], sulforaphane [172], lycopene [173], diallyl trisulfide [174], omega-3 fatty acids [175], proanthocyanidins [176], green tea polyphenols [177], genistein [178], silymarin [179], and curcumin [180,181], from plants are underway.

Table 1 :
Proposed mechanisms of phytochemicals for cancer prevention.

Table 2 :
Various assays for chemopreventive mechanisms.
use of animal efficacy models to establish organ specificity and to generate dose-response, toxicity, and other pharmacological data is a crucial component of the determinant process for chemoprevention agents.These assays with thetoxicity tests are used for decisions regarding recommendations for clinical use.Numerous animal models are used to study inhibition of chemical carcinogenesis in rodents.Important criteria considered in selecting an in vivo model for screening cancer chemoprevention agents include (i) short study duration and induction of carcinogenesis; (ii) target-specific experimental model evidenced by the induction of cancer in the target tissues comparable in such factors as histological type and hormone dependence to those

Table 3 :
Preclinical animal models for identifying chemoprevention efficacy.

Table 4 :
Transgenic/gene KO mice or rats for identifying chemoprevention efficacy.